antibiotics Article Rapid and Accurate Detection of Escherichia coli and Klebsiella pneumoniae Strains Susceptible/Resistant to Cotrimoxazole through Evaluation of Cell Elongation Isidoro López 1,2,Fátima Otero 1,2, Rebeca Guillén 1,2, María del Carmen Fernández 3, Germán Bou 3, Jaime Gosálvez 4 and José Luis Fernández 1,2,* 1 Genetics Unit, INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC), 15006 A Coruña, Spain; [email protected] (I.L.); [email protected] (F.O.); [email protected] (R.G.) 2 Molecular Genetics and Radiobiology Laboratory, Centro Oncológico de Galicia, 15009 A Coruña, Spain 3 Microbiology Division, INIBIC-Complejo Hospitalario Universitario A Coruña (CHUAC), 15006 A Coruña, Spain; [email protected] (M.d.C.F.); [email protected] (G.B.) 4 Genetics Unit, Facultad de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-981-287499; Fax: +34-981-287122 Abstract: Trimethoprim-sulfamethoxazole is a well-known antibiotic that inhibits folic acid synthesis, Citation: López, I.; Otero, F.; Guillén, a topic of renewed interest. Since resistant strains are increasingly more common, an early and R.; Fernández, M.d.C.; Bou, G.; accurate discrimination of susceptibility may assure confident therapy. Two morphological assays Gosálvez, J.; Fernández, J.L. Rapid were performed in Escherichia coli (n = 50; 27 non-susceptible) and Klebsiella pneumoniae (n = 52; and Accurate Detection of Escherichia 18 non-susceptible). First, the strains were incubated with the CLSI breakpoint of cotrimoxazole coli and Klebsiella pneumoniae Strains for 150 min, which induced cell lengthening in the susceptible strains. Second, the bacteria were Susceptible/Resistant to incubated with mitomycin C (MMC) (0.5 mg/L) for 120 min to induce a SOS-linked cell enlargement Cotrimoxazole through Evaluation of higher than that obtained by cotrimoxazole alone. When cotrimoxazole was added 30 min before Cell Elongation. Antibiotics 2021, 10, 720. https://doi.org/10.3390/ MMC, the inhibition of folic acid synthesis in the susceptible strain resulted in the suppression of antibiotics10060720 MMC-induced extra elongation. In the non-susceptible strains, folic acid synthesis continued despite the antibiotic, so that the MMC-induced extra cell lengthening could not be impeded. Whereas the Academic Editors: Seok Hoon Jeong, first assay resulted in five false negatives and four false positives of resistance, the results of the Eun-Jeong Yoon and second assay matched those of the conventional antibiogram. This simple morphological procedure Anna Giammanco is performed in 2 h and 45 min and may allow a rapid selection of useful and relatively inexpensive therapy, thereby preserving the newer broad-spectrum antibiotics. Received: 6 May 2021 Accepted: 11 June 2021 Keywords: cotrimoxazole; trimethoprim-sulfamethoxazole; antibiotic resistance; cell length; rapid Published: 15 June 2021 assay; Gram-negative Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Cotrimoxazole, the combination of trimethoprim and sulfamethoxazole, was intro- duced in the late 1960s due to their synergistic activity to block bacterial folic acid syn- thesis [1,2]. Sulfamethoxazole is a sulfonamide, structural analog of para-aminobenzoic acid that competitively inhibits dihydropteroate synthase, an enzyme that catalyzes the Copyright: © 2021 by the authors. formation of dihydrofolate from para-aminobenzoic acid and pteridine. Trimethoprim Licensee MDPI, Basel, Switzerland. This article is an open access article is a diaminopyrimidine antibiotic, structural analog of the pteridine component of dihy- distributed under the terms and drofolate, behaving as a competitive inhibitor of dihydrofolate reductase, thus blocking conditions of the Creative Commons the reduction of dyhydrofolic acid to tetrahydrofolic acid [3]. Tetrahydrofolic acid is the Attribution (CC BY) license (https:// physiologically active form of folic acid, being an essential cofactor for the synthesis of creativecommons.org/licenses/by/ thymidine, purines and methionine. The depletion of tetrahydrofolate as a consequence 4.0/). Antibiotics 2021, 10, 720. https://doi.org/10.3390/antibiotics10060720 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 720 2 of 11 of the sequential enzymatic inhibition in its metabolic pathway hinders bacterial DNA replication and transcription, as well as protein synthesis, leading to a bactericidal effect. Cotrimoxazole is usually active against Gram-negative bacilli and Gram-positive cocci but, also, Pneumocystis jirovecii and Toxoplasma gondii infections [3,4]. Nevertheless, the prescription of cotrimoxazole has been progressively declining during the past decade due to the spread of strains expressing genotypic and phenotypic resistance. In fact, cotrimoxazole remains as a first option for the treatment of noncomplicated urinary tract infections and in diseases caused by Stenotrophomonas maltophilia [5,6]. It is also prescribed for the acute infectious exacerbation of chronic obstructive pulmonary disease, otitis media in pediatric population, traveler’s diarrhea, shigellosis, pneumocystis pneumonia and toxoplasmosis [3,4]. More recently, due to the global spread of multidrug-resistant (MDR) Gram-negative bacilli, interest in the use of cotrimoxazole has been renewed. This is a widely available and relatively inexpensive medication whose adequate prescription may help to prevent the advancement of dangerous resistant pathogens by the misuse of antibiotics and to preserve newer broad-spectrum antibiotics and last option antibacterial drugs [7–9]. Since cotrimoxazole resistant strains are usual, an accurate determination of the susceptibility or resistance should be performed [10]. It is evident that the earlier the testing, the more reliable and successful the therapy will be. Trimethoprim has been shown to induce filamentation in the clinical strains of some Enterobacteriaceae, and it has been proposed as a rapid test for the discrimination of susceptibility or resistance [11]. In a previous report, we introduced a rapid assay for the identification of resistant strains of Gram-negative bacilli to antibiotic inhibitors of protein synthesis based on the simple evaluation of the bacterial cell length [12]. The cultures were incubated with mitomycin C (MMC), which induced significant cell enlargement as a rapid consequence of the triggering of the SOS response [13,14]. An addition of the antibiotic before MMC prevented the elongation when the strain was susceptible to the antibiotic, thereby inhibiting protein synthesis. However, cell enlargement was not prevented in the antibiotic-resistant strains where protein synthesis had not been successfully inhibited [12]. Here, we demonstrate that the MMC-induced elongation assay, in addition to the antibiotic inhibitors of protein synthesis, can also be successfully adapted for the rapid detection of susceptibility or resistance to cotrimoxazole in Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae. The discriminative capacity of the MMC-induced elongation assay may be more accurate than the evaluation of filamentation induced by the antibiotic. 2. Results 2.1. Technical Assay The technical bases of the assay are presented in Figure1. Two strains of E. coli, one susceptible (S) to cotrimoxazole (MIC: 0.125 mg/L) and one resistant (R) (MIC: >32 mg/L), were incubated with the CLSI (Clinical and Laboratory Standards Institute) breakpoint concentration of the antibiotic (2 mg/L) for 150 min, with MMC (0.5 mg/L) for 120 min and with cotrimoxazole 150 min and MMC added during last 120 min. Cotrimoxazole alone increased the cell length in the susceptible strain but not in the resistant strain (basal-S: 2.18 ± 0.39 µm; basal-R: 2.16 ± 0.46 µm; cotrimoxazole-S: 3.61 ± 1.19 µm; cotrimoxazole- R: 2.33 ± 0.37 µm; mean ± SD). MMC alone induced cell lengthening in both strains, higher than that produced by cotrimoxazole alone (MMC-S: 4.57 ± 2.18 µm; MMC-R: 6.49 ± 4.13 µm). When cotrimoxazole was added 30 min before MMC, the susceptible strain could not enlarge more than that achieved by cotrimoxazole alone, whereas the resis- tant strain increased the cell length similarly to that from the culture incubated with MMC alone (cotrimoxazole + MMC-S: 3.68 ± 1.35 µm; cotrimoxazole + MMC-R: 6.35 ± 4.10 µm). According to these results, two morphological parameters may provide information about the susceptibility or resistance to cotrimoxazole (1): cotrimoxazole-induced lengthening and (2) cotrimoxazole-induced suppression of MMC-linked elongation. Antibiotics 2021, 10, 720 3 of 11 Figure 1. Escherichia coli strains susceptible (left, MIC: 0.125 mg/L) and resistant (right, MIC: >32 mg/L) to cotrimoxazole. (a,b) Control without antibiotic. (c,d) Incubated with cotrimoxazole, 2 mg/L. (e,f) Treated with mitomycin C (MMC), 0.5 mg/L.(g,h) Incubated with cotrimoxazole and MMC. MMC induces cell elongation in both strains (e,f). Cotrimoxazole induces cell lengthening and prevents MMC-induced cell enlargement in the susceptible strain (c,g) but not in the resistant strain (d,h). Antibiotics 2021, 10, 720 4 of 11 2.2. Dose Response Three strains of E. coli and three of K. pneumoniae with increasing MICs of cotri- moxazole were incubated with increasing concentrations of the antibiotic, ranging from 0.06